P53 Fibril Formation and Disease

描述（由申请人提供）：我们在2002年发现，肿瘤抑制因子p53的四聚化（泰特）结构域可以从其天然构象转化为具有淀粉样蛋白原纤维特征的替代构象。此外，我们观察到，该结构域的癌症相关突变形式，其中Arg 337突变为His，与野生型结构域相比，表现出形成原纤维的显著提高的倾向，p53是结构模块化蛋白，并且泰特结构域的折叠是肿瘤抑制所需的。以前，我们发现R337 H突变以pH依赖性方式使泰特结构域不稳定，并对与此突变相关的癌症（儿童肾上腺皮质癌（ACC））的组织特异性进行了分子解释。我们认为原纤维形成和突变型p53肿瘤生物学之间存在联系。与其他癌症相关的p53突变形式的发现一致，具有R337 H突变（p53-R337 H）的p53在ACC肿瘤细胞的细胞核中以高水平积累。基于我们对突变泰特结构域的发现，我们假设这些核积累具有纤维状结构。此外，我们认为，（一些）其他突变形式的p53积累在纤维结构。我们试图通过野生型和突变型p53的多结构域形式的结构研究来测试这些假设。将应用多种生物物理学、生物化学和细胞生物学方法在体外、肿瘤细胞和来自肿瘤的培养细胞中研究p53分子。我们的目标是：1）确定野生型和突变型p53是否在体外从天然状态转化为淀粉样蛋白原纤维，和2）确定肿瘤细胞中野生型和突变型p53的聚集形式是否以原纤维状态存在。P53是一种多功能蛋白，在转录、翻译后修饰和细胞定位等多个水平上受到调控。为了了解与p53突变相关的肿瘤发生，我们必须了解这些不同的调控过程如何受到突变的影响。虽然高分辨率的结构和广泛的生物物理数据可用于几个领域，全长p53的结构和生物物理特性仍然是一个谜。证明突变型p53分子作为原纤维存在将解释报道的p53积累及其核定位的高稳定性，将为功能丧失提供分子解释，并将为新型p53定向治疗的发展提供见解。

DESCRIPTION (provided by applicant): We discovered in 2002 that the tetramerization (tet) domain of the tumor suppressor p53 can be converted from its native conformation to an alternative conformation with the characteristics of amyloid fibrils. Further, we observed that a cancer-associated mutant form of this domain, with Arg 337 mutated to His, exhibits a significantly heightened propensity to form fibrils compared to the wild-type domain, p53 is a structurally modular protein, and folding of the tet domain is required for tumor suppression. Previously, we showed that the R337H mutation destabilizes the tet domain in a pH-dependent manner and developed a molecular explanation for the tissue specificity of cancer associated with this mutation (adrenal cortical carcinoma (ACC) in children). We feel that there is a link between fibril formation and mutant p53 tumor biology. Consistent with findings for other cancer-associated mutant forms of p53, p53 with the R337H mutation (p53-R337H) accumulates at high levels in the nuclei of ACC tumor cells. Based on our findings for the mutant tet domain, we hypothesize that these nuclear accumulations have fibrilar structure. Further, we suggest that (some) other mutant forms of p53 accumulate in fibrilar structures. We seek to test these hypotheses through structural studies of multi-domain forms of wild-type and mutant p53. A variety of biophysical, biochemical and cell biology methods will be applied to study p53 molecules in vitro, in tumor cells, and in cultured cells derived from tumors. Our aims are: 1) To determine whether wild-type and mutant p53 convert from the native state to amyloid fibrils in vitro, and 2) To determine whether aggregated forms of wild-type and mutant p53 in tumor cells exist in a fibrilar state. P53 is a multifunctional protein that is regulated at many levels, including transcription, post-translational modification and cellular localization. To understand tumorigenesis associated with mutations to p53, we must understand how these different regulatory processes are affected by mutations. While high-resolution structures and extensive biophysical data are available for several domains, the structural and biophysical properties of full-length p53 remain largely a mystery. Demonstrating that mutant p53 molecules exist as fibrils would explain the high stability reported for p53 accumulations and their nuclear localization, would provide a molecular explanation for loss of function, and would offer insights into the development of novel p53-directed therapeutics.